Diagnostic testing of infectious and sexually transmitted diseases has become increasingly focused on faster, more accurate results. Nucleic acid probe technology has enabled rapid diagnostic testing to break time-to-result barriers with high specificity, and less subjectivity. While they are faster than growth based, biochemical assays, and more specific than immunologically based assays, nucleic acid probe assays present a unique challenge for delivering the target sample intact. Furthermore, samples that may be collected at one site, and tested at another site, are particularly vulnerable to nucleic acid degradation if not handled properly.
Nucleic acid detection by hybridization and capture has been applied to a host of diseases. It has been especially useful for infectious diseases in which conventional methods are time consuming, expediency of treatment is critical, and/or the disease is reportable to health agencies. Trichomonas vaginalis vaginitis (trichomoniasis) is a reportable sexually transmitted disease that affects approximately 3 million women per year in the U.S. Furthermore, vaginal disorders due to bacterial vaginosis (BV) and candidiasis, are two of the most common reasons women seek medical treatment. The symptoms of these three distinct diseases overlap, thus creating a need for differential diagnosis before appropriate and specific medication can be prescribed. A rapid and accurate diagnosis is especially critical in pregnant women, in whom BV and trichomoniasis are associated with premature births and low birth weight babies. Moreover, BV-positive pregnant women are predisposed to chorioamnionitis, amniotic fluid infection, and puerperal infectious morbidity. BV has also been associated with pelvic inflammatory disease, postpartum endometritis, bacteremia, salpingitis, and the like. Proper diagnosis and treatment of vaginitis requires identifying the causative microorganism so that the appropriate antimicrobial treatment can be defined.
The Affirm VPIII nucleic acid hybridization assay, described in U.S. Pat. No. 5,654,418, is a significant advance in the diagnosis of vaginitis, due to its ability to detect T vaginalis, G. vaginalis or C. albicans, from a single vaginal swab. The swab is incubated in a lysis solution at high temperature, which causes the organism to lyse and release nucleic acid. A buffer solution is then added to the sample. The sample solution is next incubated with a set of nylon beads that are each derivatized with specific capture probes. The rRNA hybridizes to the capture beads, which are next incubated with a solution containing biotinylated detector oligonucleotide probes. The detector probes hybridize to another region in the rRNA. The bead is transferred to a well containing an enzyme. If biotinylated detector probes are hybridized to the rRNA, the enzyme will bind to the biotin. If there is no rRNA hybridized to the bead, no biotinylated detector probe will be present for the enzyme to bind. Finally, the beads are incubated with a substrate, which will react with the enzyme to form a blue color. If rRNA is present the beads will appear blue. If there is no rRNA in the sample, the beads remain colorless. A differential diagnosis can be obtained from a single sample by using three beads, each bead specific for only one of the analytes.
The present invention was developed to provide stability to vaginal swab samples, specifically, samples collected to test for the presence of Candida, Gardnerella or Trichomonas, using the Affirm VPIII Microbial Identification Test. Without the aid of the present invention, the swab samples will only remain stable for up to one hour at ambient temperature, or four hours at refrigerated temperature. Specifically, the rRNA within the cells must remain intact in order to be detected, and, the presence of low, non-pathological numbers of Candida must be kept from multiplying and producing a false positive. The proper preservative would allow for sample collection and sample testing to be conducted at remote sites, or for numbers of samples to be batched for processing and testing all at once.
The appropriate transport or preservative or fixative solution for the Affirm VPIII sample needed to have the following attributes:                1. The solution had to control or inhibit RNA degrading enzymes (RNases) found in vaginal fluid.        2. It had to prevent growth of Candida, Gardnerella or Trichomonas, while . . .        3. controlling RNA degradation within the cells due to endogenous nucleases or cell death.        4. The solution had to be compatible with the Affirm VPIII test as embodied.        5. It could not introduce unnecessary risk to the end users, and        6. It would provide signal stability for samples stored up to 72 hours.        
Conventional preservatives, such as those having bactericidal or inhibitory effects, prevent growth of low levels of organisms, but do not address the problem of nucleic acid degradation. Conversely, transport media tend to be minimal or starvation media, formulated to maintain viability of the organism for culture later. Candida, however, tend to flourish in such media, while Gardnerella and Trichomonas do not survive. The result with such media is a false positive Affirm result for Candida and a false negative for the latter two. The complexity of the problem is increased by the fact that these three organisms represent both prokaryotic and eukaryotic cell types, and each has a distinctly constructed cell wall and/or membrane.
Fixatives, as a class of substances, tend to contain alcohol, formaldehyde or chloroform, and a wide range of additives, depending on the specimen and application. Many are not stable solutions, suitable only for use within hours of preparation. Furthermore, they may present hazards beyond those already faced by the clinician (i.e., mercuric chloride, picric acid). Formaldehydes were found to be incompatible with Affirm reagents. Alcohol based fixing agents offered the most promise due to their ability to precipitate or denature proteins, particularly, nucleases.
One such fixative is described in U.S. Pat. No. 5,256,571, for preserving the structure of mammalian cells. Hurley et al claim a solution (designated from here on as PreservCyt) of 45 to 55% methanol, an anti-clumping agent and a buffering agent. However effective this solution may be for mammalian cells, it was not capable of meeting the criteria outlined above for all three vaginal pathogens of interest. This was most likely due to the added complexity of the cell wall structure of each organism, a structure not encountered in mammalian cells. Furthermore, in these studies, increasing the methanol concentration to 95% did not preserve, or fix, the vaginal samples such that the rRNA was detectable after 24 hours.
Many fixatives used for cytology and histology are home-brewed solutions, the formulations of which are well known to those skilled in the art. They are frequently prepared fresh and used within a short period of time, as mentioned above. Such solutions may include for example: 10% neutral buffered formalin, Carnoy's solution (ethanol, chloroform, acetic acid), B-5 (mercuric chloride, sodium acetate, formalin, water), Bouin's solution (picric acid, glacial acetic acid, formaldehyde), and Zenker's solution (water, potassium dichromate, mercuric chloride, glacial acetic acid). These formulations are routinely published in reference pages, via the internet, by academic or research institutions such as: the University of Bristol's Department of Pathology & Microbiology (lang-dl-srv.lang.bris.ac.uk/Cpl/histfix.htm), The Jackson Laboratory (jax.org/resources/documents/sss/imaging/histf.html), and the University of Newcastle at Australia (newcastle.edu.au/department/bi/birjt/techinfo/bio_fix.html). One lesser known fixative, published via the internet by the University of Texas,Austin (vize222.zo.utexas.edu/Marker_pages/methods_pages/fixatives.html), is known as Dents solution. This solution contains four parts methanol and one part dimethyl sulfoxide (DMSO). Dents solution is described as a fixative used for immunological staining of Xenopus specimens. The specified protocol is to fix samples at −20° C. overnight. The authors claim that samples prepared in this manner may be stable frozen for many months, perhaps years.
It has been found by the inventors that the above described Dents solution will preserve vaginal swab samples containing vaginal fluid and seeded quantities of Trichomonas vaginalis, Gardnerella vaginalis, and Candida albicans. Swabs stored in this solution are stable for several days at ambient temperature, prior to testing in the Affirm VPIII system. Results obtained with such swabs give similar signals to identical swabs tested immediately after preparation. It has also been found by the inventors that ethanol, or mixtures of ethanol and methanol, will also preserve the samples when mixed with DMSO. The preferred embodiment uses a 1:1 mix of methanol and DMSO.
Dimethyl sulfoxide has been used as an ingredient mixed with other molecules in previous references but in significantly smaller concentrations. U.S. Pat. No. 5,622,867 uses 0.5 to 6% DMSO in a solution to store blood platelets; U.S. Pat. No. 3,852,155 uses 8 to 10% DMSO in a solution for cryopreserving equine cell cultures; U.S. Pat. No. 5,364,756 describes a solution containing 0.5M (˜3-4%) DMSO. U.S. Pat. Nos. 5,422,277 and 4,666,699 use DMSO in stain-fixative solutions at 5 to 10% and 3 to 8% respectively. However, in every one of these instances, DMSO is only a small fractional component of a much more complex solution.
In the present invention, the inventors describe a novel composition which is a mixture of a first substance which is at least one alcohol or ketone and a second facilitating substance such as DMSO, with a preferred embodiment being a mixture of 50% methanol/50% DMSO, and the method of providing long term (several days) stability, for example, to cells and in particular, clinical specimens, utilizing this composition.
Specifically, in a preferred embodiment, the clinical specimens are considered to be vaginal swabs, containing the causative agents for vaginitis and bacterial vaginosis, however, the solution could be used for other biological specimens in which the recovery of RNA is necessary. This solution will be useful in preventing the degradation of nucleic acids (i.e., DNA, RNA) located within cells, suspended in a matrix of biological fluid such as vaginal fluid. Furthermore, this solution will be capable of preventing degradation of, for example, RNA, an easily degraded nucleic acid, over several days at ambient temperatures and above.